Living cells suspended in fluid growth medium, for example in a bioreactor, have been used to generate pharmaceutically useful molecules. In many cases, the molecules produced by the cells are discharged into the growth medium; in other cases, the product is within the cells or may constitute the cells themselves; simultaneously, in search of increased productivity, the practice of cell culture has evolved. In one culture method, cells are grown in a continuous manner and to high concentrations by removing waste products from the culture and replacing with fresh media. In many cases, therefore, separation of cells from growth medium becomes an essential step in production of cell derived products. Separation of the molecules from the particulate cells in suspension or attached to microcarriers suspended in the growth medium can be achieved by a variety of methods. Not excluding other suspensions or solutions, the focus going forth will be on the use of anchorage dependent cells cultured on microcarriers. One separation method involves a “screen cage” with a mesh of pore size smaller than the microcarriers. The cage, in many cases, is placed in the culture vessel itself, and when appropriate, can be used to separate microcarriers from suspending medium, retaining the microcarriers within the vessel. The screen cage, while used in numerous processes, has a number of flaws, including that it is prone to clogging. Once clogged, it becomes useless and may result in premature termination of the production run at great cost and time loss. Furthermore, the volume of the internal screen cage reduces the capacity of the culture production vessel.
Another method, somewhat like an external screen cage or using a chamber and a partitioning screen, has been used for separation of microcarriers from culture medium. It involves continuous pumping of culture suspension through the screen. The screen retains the microcarriers and the media flows through. This device results in concentration of microcarriers within the separation chamber; however, while effective for short separation steps, it may result in entrapment of the microcarriers within the screened chamber causing its eventual clogging. Another limitation of this method, inherently results from the concentration of microcarriers with attached cells within the chamber during the separation process, a process that can deprive the cells of essential nutrients and lead to cell damage.
Another method for separating microcarriers from a culture medium involves a settling process, involving the of use of microcarriers, with attached cells, that together are heavier than the suspending medium. In a static culture, without agitation, the microcarriers, which are of specific gravity greater than the suspending medium, will settle to the bottom of the culture vessel, allowing removal of microcarrier-free medium from the top. While this method is reliable and commonly used, it is not preferred. The settling process is slow and time consuming, particularly at large scale, where settling distances are great. In addition, maintaining the cells in an unagitated environment can deprive the cells of oxygen and other nutrients. The current invention is designed to alleviate some of the limitations of other current systems.
The prior art provides filters that allows the molecules, but not larger particulate matter or cells to pass through it. In order to maximize the production of the molecules, systems have been developed to replenish the medium removed from the suspended entities during the filtration step. This has been achieved in the prior art using alternating tangential flow systems (See U.S. Pat. No. 6,544,424). The system described in that patent, however, are not well adapted to disposability nor does it provide a mechanism for controlling the flow dynamics across the filter surface that may enhance the capacity and efficiency of the filter. The use of a device that can controls the flow dynamics or patterns across the filter membrane may be used to enhance the effectiveness of the filter. The term filter includes, but is not limited to, any of ultrafiltration filters microfiltration filters, macrofiltration filters as well as screens. The ability to control the flow dynamics across a screen filter facilitates its use, as exemplified, in production of vaccines, a multistep process; examples of the steps include an initial wash of microcarriers, meaning rapid removal suspending media through the screen filter and retaining the microcarriers and replenishing removed media with fresh media. Such step may be repeated more than once; another step, follows steam sterilization of the suspended microcarriers, which also requires a rapid media exchange step, removing sterilization media and replacement with fresh growth media, so that the subsequent inoculation with cells will result in rapid attachment and growth of the cells on the microcarriers; a further step may include removal of growth media from the culture, retaining microcarriers and attached cells, followed by addition of a second, production, media and simultaneously inoculation with a virus; following viral growth phase, the virus laden cells may result in cell lysis; in which case, the screen filter may be used to separate and harvest the virus, retaining the microcarriers and cell remnants in the culture vessel. Facilitation of such multistep process by an efficient separation device such as described by the invention can greatly enhance the viral production process and making the process more efficient, reliable and cost effective. it would be desirable to have a less expensive system, preferably one that could be considered disposable. A disposable system would not have to be washed or prepared for use, time consuming efforts that decrease system reliability and increase operating costs It would also be simpler to dispose of and replace a spent system by an unused filter module as needed.
The present invention provides an enhanced screen filter module that can be used in a disposable manner if desired and an enhanced means for controlling the flow dynamics across a filter to enhance its filtration capacity and its usefulness in a greater range of applications.